IBC-1 (Invasive Breast Cancer-1), a putative oncogene amplified in breast cancer

ABSTRACT

This invention encompasses antibodies specific for IBC-1 (Invasive Breast Cancer-1), methods for diagnosis and prognosis of metastatic breast cancer and degenerative neural conditions, methods of identifying and manufacturing therapeutic compounds, and methods of treating patients with invasive and metastatic breast cancer or degenerative neural conditions.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part, and claims priority,of International Application No. PCT/US02/34449, filed Oct. 28, 2002,which claims priority of U.S. Provisional Application Serial No.60/343,154, filed Oct. 26, 2001, and U.S. Provisional Application SerialNo. 60/356,301, filed Feb. 12, 2002, the contents of all of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] This invention relates to cancer and neurodegenerative diseases.

BACKGROUND

[0003] Breast cancer is a leading cause of cancer deaths in womenworldwide. Despite recent improvements in cancer therapy, advanced stagetumors are still almost inevitably fatal (Alberg et al. (2000) Curr OpinOncol 12:515-520). Therefore, there is a need for the identification ofnovel therapeutic targets, particularly in estrogen receptor negativeand metastatic tumors which are the least responsive to currenttherapies.

SUMMARY

[0004] This invention is based on identification of a human gene that isexpressed in an aggressive subset of invasive breast carcinomas and inthe pons of the brain, but not in 75 other normal human adult and fetaltissues. This gene was designated IBC-1 (Invasive Breast Cancer-1). Thepredicted amino acid sequence encoded by IBC-1 cDNA contains sequencesimilar or identical to that of (a) a previously identified humancachexia-associated protein (Akerblom et al., U.S. Pat. No. 5,834,192),(b) a protein fragment derived from PIF (Proteolysis Inducing Factor;also called cancer cachexia factor), (c) a putative secreted neuralsurvival peptide (Cunningham et al. (1998) J Neurosci 18:7047-7060); andTodorov et al. (1996) Nature 379:739-742), and (d) a dermcidin proteinexpressed in sweat glands of the skin (Schittek et al. (2001) NatureImmunology 2:1133-1137). IBC-1 encodes a 110 amino acid pro-protein(i.e., “pro-IBC-1;” SEQ ID NO:3) with a predicted 19 amino acid signalpeptide at the N-terminal, which is presumably removed upon maturationof the protein.

[0005] As used herein, “pro-IBC-1” refers to the 110 amino acid proteinwith a putative signal peptide, whereas “IBC-1” refers to the 91 aminoacid mature protein (i.e., amino acids 20-110 of pro-IBC-1; SEQ IDNO:4).

[0006] This invention relates to antibodies specifically binding toIBC-1, methods of diagnosing and prognosticating cancer and neuraldiseases, methods of identifying and manufacturing a therapeuticcompound, and methods of treating cancer and neural diseases. The cancercan be, for example, a breast cancer, pancreatic cancer, brain cancer,gastric cancer, lung cancer, lymphoma, or any other type of cancer thatis invasive and metastatic and that may or may not be associated withcachexia.

[0007] More specifically, this invention includes a purified antibodythat specifically binds to an epitope within a fragment of IBC-1, e.g.,within (or in) a fragment that includes or consists of the sequence of:SEQ ID NO:5 (amino acids 20-42 of pro-IBC-1); SEQ ID NO:6 (amino acids43-64 of pro-IBC-1); SEQ ID NO:7 (amino acids 53-64 of pro-IBC-1); SEQID NO:12 (amino acids 45-64 of pro-IBC-1); or SEQ ID NO:13 (amino acids86-103 of pro-IBC-1). The antibody can be a monoclonal antibody or apolyclonal antibody. These antibodies can be used for detecting IBC-1 ina test sample from a patient, identifying a therapeutic compound, andtreating diseases associated with overexpression of IBC-1.

[0008] As used herein, the term “antibody” refers not only to wholeantibody molecules, but also to antigen-binding fragments, e.g., Fab,F(ab′)₂, Fv, and single chain Fv (ScFv) fragments. Also included arechimeric antibodies, such as humanized antibodies.

[0009] The term “purified antibody,” as used herein, refers to anantibody which either has no naturally-occurring counterpart or has beenseparated from components which naturally accompany it, e.g., bloodcells.

[0010] Also within the invention are methods of diagnosis and prognosis.One such method is based on determining whether a test sample contains areceptor for IBC-1. The method involves: (a) providing a test samplefrom a human patient; (b) contacting the test sample with a polypeptidecontaining 10-91 consecutive amino acids of IBC-1; and (c) determiningwhether the polypeptide binds to the test sample. Detection of thepolypeptide bound to the test sample in an amount higher than a negativecontrol indicates that the test sample contains a receptor for IBC-1.The polypeptide can be, for example, IBC-1 itself, or a fragment ofIBC-1, 10 to 91 amino acids in length (e.g., 10 to 50 amino acids,preferably 12 to 40 amino acids, and more preferably 15 to 30 aminoacids in length). The test sample can be prepared from a breast cancertissue sample if the patient is suspected of having, or being likely todevelop, invasive and metastatic breast cancer. In such a case, thepresence of the receptor in the test sample in an amount higher than acontrol sample indicates that the patient has, or is likely to develop,invasive and metastatic breast cancer. If the patient is suspected ofsuffering from, or being at risk for developing, a degenerative neuralcondition, the test sample can be prepared from a brain tissue sample,such as a substantia nigra, pons, or hypothalamus tissue sample. In sucha case, an amount of the receptor in the test sample less than an amountof the receptor in a sample from a normal human (i.e., a person withoutany degenerative neural condition) indicates that the patient issuffering from, or at risk for developing, a neural condition involvingdegeneration of substantia nigra, pons or hypothalamus cells, or otherneural cells normally protected by the presence of IBC-1.

[0011] Another method of diagnosis and prognosis is based on determiningwhether a test sample contains the IBC-1 mRNA or IBC-1. The methodinvolves: (a) providing a test sample from a patient; and (b) detectingIBC-1 rMRNA or IBC-1 in the test sample. If the patient is suspected ofhaving, or being likely to develop, invasive and metastatic breastcancer, the test sample can be prepared from a breast cancer tissuesample, or a body fluid (e.g., urine, breast milk, saliva, or blood);the presence of a higher than control level of IBC-1 mRNA or IBC-1 inthe test sample indicates that the patient has, or is likely to develop,invasive and metastatic breast cancer. If the patient is suspected ofsuffering from, or being at risk for developing, a degenerative neuralcondition, the test sample is typically prepared from a substantianigra, pons or hypothalamus tissue sample, or from a body fluid (e.g.,urine, cerebro-spinal fluid, saliva, or blood). An amount of IBC-1 MRNAor IBC-1 in the test sample less than an amount of IBC-1 MRNA or IBC-1in a comparable sample from a normal human indicates that the patient issuffering from, or at risk for developing, a neural condition involvingdegeneration of substantia nigra, pons or hypothalamus cells, or othertypes of neural cells that normally express IBC-1.

[0012] A third method of diagnosis and prognosis is based on determiningwhether genomic IBC-1 DNA is amplified in a test sample. The methodinvolves: (a) providing a test sample comprising genomic DNA from abreast cancer patient; and (b) determining whether genomic IBC-1 DNA isamplified in the test sample. If the patient is suspected of having, orbeing likely to develop, invasive and metastatic breast cancer, the testsample can be prepared from a breast cancer tissue sample; the presenceof amplified genomic IBC-1 DNA in the test sample indicates that thepatient has, or is likely to develop, invasive and metastatic breastcancer. As used herein, “amplified” means that the amount of genomic DNAsequences in a cell that can be transcribed into mRNA molecules thatencode functional IBC-1 protein molecules is higher than that in acontrol person (e.g., a person without breast cancer).

[0013] Also included in this invention is a method for identifying acompound that blocks binding of IBC-1 to its receptor. The methodinvolves: (a) providing a polypeptide that contains between 10 and 91consecutive amino acids of IBC-1 and binds an IBC-1 receptor; (b)providing a cell expressing the IBC-1 receptor; (c) contacting the cellwith the polypeptide in the presence of a test compound; and (d)determining whether the test compound blocks binding of the polypeptideto the cell, as an indication that the compound blocks binding of IBC-1to its receptor. The polypeptide can be, for example, IBC-1 itself, or afragment thereof at least 10 amino acids in length (preferably at least12 amino acids, more preferably at least 15 amino acids, e.g., at least20 or at least 50 amino acids in length). Useful IBC-1 fragments can beany IBC-1 fragments disclosed herein. The cell can be a breast cancercell (e.g., an invasive breast cancer cell) or a neural cell (e.g., asubstantia nigra, pons or hypothalamus cell), or any other cell thatexpresses the receptor. The test compound can be, for example, apeptide, a non-peptide small molecule, or an antibody that binds toIBC-1 or its receptor. A compound thus identified can be used fortreating diseases associated with overexpression of IBC-1. If it blocksby binding to IBC-1, it can also be used for detecting IBC-1 in a sample(e.g., for diagnosis and prognosis as described above). If it blocks bybinding to the IBC-1 receptor, it can also be used to detect thepresence of the receptor on a cell (e.g., for diagnosis and prognosis asdescribed above). Once a compound that blocks binding of IBC-1 to itsreceptor has been identified, it can be manufactured in a large scale.

[0014] In another aspect, this invention provides a method of treatingcancer. The method involves: (a) identifying a patient having, or beinglikely to develop, an invasive and metastatic breast cancer thatexpresses IBC-1 or an IBC-1 receptor; and (b) treating the patient with(i) a compound that blocks binding of IBC-1 to its receptor (e.g., anon-agonistic antibody that binds to IBC-1 or its receptor) or (ii) acompound that inhibits expression of IBC-1 or its receptor (e.g., anRNAi molecule). The patient may or may not be diagnosed as sufferingfrom cachexia, or exhibiting overt symptoms of cachexia (e.g.,unintentional loss of at least 10% of body weight in a short period oftime).

[0015] Yet another aspect of this invention is a method of treating aneural condition by the steps of (a) identifying a patient sufferingfrom, or at risk for developing, a neural condition involvingdegeneration of substantia nigra, pons or hypothalamus cells, or anothertype of neural cell that normally expresses IBC-1; and (b) administeringIBC-1 or an IBC-1 agonist to the patient.

[0016] Also within the scope of the invention is a kit for detectinginvasive and metastatic breast cancer. The kit comprises (a) an agentfor determining the level of IBC-1 in a biological sample, or (b) anagent for determining whether genomic IBC-1 DNA is amplified in abiological sample; and instructions for use of the agent for detectinginvasive and metastatic breast cancer.

[0017] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. In case of conflict,the present document, including definitions, will control. Preferredmethods and materials are described below, although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention. All publications,patent applications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting. Other features, objects, and advantages of the invention willbe apparent from the description and the accompanying drawings, and fromthe claims.

DESCRIPTION OF DRAWINGS

[0018]FIG. 1A is a diagram of the genomic structure of the IBC-1 gene.Exon-intron boundaries, start and stop codons, and the SAGE tag and theadjacent NlaIII site (SEQ ID NO:8) that led to the identification ofIBC-1 are indicated.

[0019]FIG. 1B is a representation of the nucleic acid sequence of a cDNA(SEQ ID NO:1) encoding pro-IBC-1 and the predicted amino acid sequence(SEQ ID NO:3) of pro-IBC-1. Sequences of peptides derived from acachectic factor and a neural survival peptide are indicated by thickand thin underlines, respectively. A predicted secretory signalpeptidase cleavage site is marked by an arrow.

[0020]FIG. 1C is an amino acid sequence alignment of IBC-1 (i.e.,pro-IBC-1; SEQ ID NO:3), lacritin (SEQ ID NO:10), and EST-AI12471 (SEQID NO:11) proteins. Amino acids identical to the consensus are shaded.Comparison was made using DNAStar and the Clustal algorithm.

[0021]FIG. 2A is a schematic representation of controlalkaline-phosphatase (AP) protein, alkaline-phosphatase-IBC-1 fusion(AP-IBC-1) protein, and binding of AP-IBC-1 ligand to a hypotheticalmembrane protein.

[0022]FIG. 2B is a bar graph depicting relative levels of AP activitybound to various human cell lines in the form of AP-IBC-1 protein. Celllines are listed on the x-axis, while the y-axis indicates boundalkaline phosphatase activity expressed as OD/hour/ml. “G361+HID-5” and“G361+IBC-1” on the x-axis indicate that G361 cells were pre-incubatedwith purified recombinant HID-5/psoriasin (1.93 mM) and IBC-1 (0.567mM), respectively, prior to AP-IBC-1 (25 nM) binding. Decreased AP-IBC-1binding in the presence of IBC-1, but not in the presence ofHID-5/psoriasin, indicates that the binding is specific for IBC-1.

[0023]FIG. 2C is a line graph illustrating binding of AP-IBC-1 to G361cells over various concentrations of AP-IBC-1. The insert showsScatchard transformation.

DETAILED DESCRIPTION

[0024] This invention is based on the identification andcharacterization of a gene encoding IBC-1. IBC-1 was identified as aSAGE tag with no match in the Unigene and Genbank databases. It wasrepresented only in those SAGE libraries that were generated frominvasive and metastatic breast carcinomas. Subsequent analyses revealedthat the predicted amino acid sequence of the IBC-1 protein containssequences identical to a previously identified human cachexia-associatedprotein, a tryptic peptide derived from a previously describedproteolysis/cachexia inducing factor (PIF), a neuronal survival peptide,and a dermcidin protein.

[0025] In the panel of breast tumors studied, all tumors that expressedIBC-1 were found to be high-nuclear grade and overexpressing erbB2.Tumors with these characteristics have a poor clinical prognosis, andare less common in postmenopausal women. Importantly, primary tumors inwhich IBC-1 protein was detected were more likely to be stage 2 or stage3 breast cancers than stage 1 breast cancer. Tumor stage is determinedaccording to the summary of 3 scores that are given based on tumors size(T), lymph nodes (N), and distant metastasis (M). Stage 1 tumors areTIN0M0 tumors. Stage 2 tumors are either large invasive tumors withoutlymph nodes and distant metastases (T2N0M0) or small invasive tumorswith lymph nodes (TIN1M0). Stage 3 tumors are primary invasive tumorswith lymph nodes but no distant metastases (T2-3N1-2M0). Therefore,expression of IBC-1 defines a clinically relevant sub-group of tumorsand represents a new therapeutic target for the treatment of thesetumors as well as cancer-associated cachexia.

[0026] In addition to a subset of invasive breast carcinomas, IBC-1 isalso expressed in the pons and paracentral gyrus of the brain, but notin any other normal adult or fetal tissues tested. The restrictedexpression pattern makes IBC-1 a good candidate cancer diagnostic markerand therapeutic target. The secreted nature and extracellular mechanismof IBC-1 action make it even more attractive for such potential uses.Consistent with this, the inventors demonstrated the existence of a cellsurface IBC-1 binding protein (i.e., putative IBC-1 receptor) in breastcancer and neuronal cells in vivo. In addition, tumors that expressIBC-1 appear to have more, or higher affinity, IBC-1 binding proteins onthe cell surface, which can further facilitate the potential therapeutictargeting of the IBC-1 pathway in these cells.

[0027] Previous studies showed that a 30 amino acid peptidecorresponding to a portion of the predicted IBC-1 amino acid sequenceappears to protect cells from oxidative insult-induced apoptosis(Cunningham et al. (1998) J Neurosci 18:7047-7060). Neurons areparticularly sensitive to reactive oxygen species (ROS), whereas tumorcells themselves produce large amounts of ROS (Szatrowski and Nathan(1991) Cancer Res 51:794-798). Therefore, the high expression of IBC-1in these cell types is likely to be essential for their survival.

[0028] Interestingly, all the neurons that strongly bind IBC-1 in thebrain play a direct or indirect role in the regulation of energyhomeostasis. Noradrenergic neurons of the locus ceruleus haveprojections to all major parts of the brain and spinal cord, and areinvolved in maintaining vigilance (arousal) status. Similarly,dopaminergic neurons of the substantia nigra are connected to thecortex, spinal cord, and hypothalamus, and regulate initiativebehavioral responses. Finally, and most interestingly, the Jateralhypothalamus is thought to be a “feeding center” and damaging itsneurons leads to impaired food intake (Inui (1999) Cancer Res59:4493-4501). Strong cell surface binding of IBC-1 to these cellssupports a role for IBC-1 in regulating feeding behavior.

[0029] In addition, catecholaminergic (noradrenergic and dopaminergic)neurons are particularly susceptible to oxidative stress, since thebiosynthesis of these neurotransmitters from tyrosine requires molecularoxygen. Moreover, the auto-oxidization of catecholamines, the endproduct of which is melanin that accumulates in neurons of thesubstantia nigra and locus ceruleus, leads to the generation of ROS(H₂O₂, O₂ ⁻, and OH). The strong binding of IBC-1 to these neurons isconsistent with its putative role as a neural survival factor thatprotects against oxidative stress. In contrast to its low and restrictednormal expression pattern, the aberrant overexpression of IBC-1 bycertain carcinomas leads to elevated circulating IBC-1 protein levels(Wigmore et al. (2000) Br J Surg 87:53-58; and Cabal-Manzano et al.(2001) Br J Cancer 84:1599-1601). Due to its small size, the IBC-1protein is able to cross the blood-brain barrier (Cunningham et al.(1998) J Neurosci 18:7047-7060). Therefore, elevated systemic IBC-1levels increase the binding of IBC-1 to neurons of the pons, midbrain,and hypothalamus, resulting in altered feeding behavior that incombination with increased muscle wasting leads to cancer related weightloss. Tumors of breast cancer patients with cachexia were found to bemore resistant to chemotherapy than those of patients withoutsignificant weight loss (Dewys et al. (1980) Am J Med 69:491-497),consistent with IBC-1 playing a role in the regulation of breast cancercell survival and feeding behavior.

[0030] IBC-1 Antibodies

[0031] This invention features antibodies that bind to an epitope withina fragment of the IBC-1 protein, e.g., SEQ ID NO:5(YDPEAASAPGSGNPCHEASAAQK, amino acids 20-42 of pro-IBC-1), SEQ ID NO:6(ENAGEDPGLARQAPKPRKQRSS, amino acids 43-64 of pro-IBC-1), SEQ ID NO:7(RQAPKPRKQRSS, amino acids 53-64 of pro-IBC-1), SEQ ID NO:12(AGEDPGLARQAPKPRKQRSS, amino acids 45-64 of pro-IBC-1), or SEQ ID NO:13(DAVEDLESVGKGAVHDVK, amino acids 86-103 of pro-IBC-1). These fragmentsare predicted to be antigenic and localized on the surface of theprotein by analysis using MacVector, and thus particularly useful ingenerating IBC-1 antibodies.

[0032] Such antibodies can be polyclonal antibodies derived from theserum or plasma of animals (e.g., mice, hamsters, gerbils, rabbits,rats, guinea pigs, sheep, horses, goats, cows, or pigs) that have beenimmunized with intact IBC-1 or a portion thereof containing the relevantIBC-1 epitope using methods, and optionally adjuvants, known in the art.Such polyclonal antibodies can be isolated from serum or plasma bymethods known in the art.

[0033] Monoclonal antibodies that bind to the above IBC-1 fragments arealso encompassed by the invention. Methods of making and screeningmonoclonal antibodies are well known in the art.

[0034] Once the desired antibody-producing hybridoma has been selectedand cloned, the resultant antibody can be produced by a number ofmethods known in the art. For example, the hybridoma can be cultured invitro in a suitable medium for a suitable length of time, followed bythe recovery of the desired antibody from the supernatant. The length ofculture time and medium are known or can be readily determined.

[0035] Additionally, recombinant antibodies specific for an IBC-1fragment described above, such as chimeric and humanized monoclonalantibodies comprising both human and non-human portions, are within thescope of the invention. Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example, using methods described in Akira et al., EuropeanPatent Application 184,187; Taniguchi, European Patent Application171,496; Morrison et al., European Patent Application 173,494; Neubergeret al., WO 86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly etal., European Patent Application 125,023; Better et al. (1988) Science240:1041-43; Liu et al. (1987) J Immunol 139:3521-26; Sun et al. (1987)PNAS USA 84:214-18; Nishimura et al. (1987) Canc Res 47:999-1005; Woodet al. (1985) Nature 314:446-49; Shaw et al. (1988) J Natl Cancer Inst80:1553-59; Morrison (1985) Science 229:1202-1207; Qi et al. (1986)BioTechniques 4:214; Winter, U.S. Pat. No. 5,225,539; Veroeyan et al.(1988) Science 239:1534; and Beidler et al. (1988) J Immunol141:4053-60.

[0036] Also included within the scope of the invention are antibodyfragments and derivatives that contain at least the functional portionof the antigen binding domain of an antibody that binds specifically toan IBC-1 fragment described above. Antibody fragments that contain thebinding domain of the molecule can be generated by known techniques. Forexample, such fragments include, but are not limited to: F(ab′)₂fragments that can be produced by pepsin digestion of antibodymolecules; Fab fragments that can be generated by reducing the disulfidebridges of F(ab′)₂ fragments; and Fab fragments that can be generated bytreating antibody molecules with papain and a reducing agent. See, e.g.,National Institutes of Health, 1 Current Protocols In Immunology,Coligan et al., ed. 2.8 and 2.10 (Wiley Interscience, 1991). Antibodyfragments also include Fv (e.g., single chain Fv (scFv)) fragments,i.e., antibody products in which there are few or no constant regionamino acid residues. An ScFv fragment is a single polypeptide chain thatincludes both the heavy and light chain variable regions of the antibodyfrom which the ScFv is derived. Such fragments can be produced, forexample, as described in U.S. Pat. No. 4,642,334, which is incorporatedherein by reference in its entirety.

[0037] Methods of Diagnosis and Prognosis

[0038] This invention also features diagnostic and prognostic assays.Such assays are based on the findings that: (a) the IBC-1 gene isexpressed only in the tumors of an aggressive subset of breastcarcinomas and in the pons, hypothalamus, and midbrain of the brain; (b)the IBC-1 gene is amplified in the same breast tumors where the IBC-1gene is expressed; and (c) there is evidence for the existence of a cellsurface IBC-1 binding protein (i.e., IBC-1 receptor) in cells whereIBC-1 is expressed. Thus, detections of either (a) IBC-1 mRNA or IBC-1in a breast cancer tissue sample or a body fluid (e.g., urine or blood)in an amount higher than in a control sample, (b) amplified genomicIBC-1 DNA in a breast cancer tissue sample, or (c) IBC-1 receptor in abreast cancer tissue sample in an amount higher than a control sample,would indicate that the patient has, or is likely to develop, invasiveand metastatic breast cancer. Control samples are preferably from normalsubjects, i.e., subjects without breast cancer. However, they can alsobe from patients with ductal carcinoma in situ (DCIS). Detection ofeither (a) IBC-1 mRNA or IBC-1 in a substantia nigra, pons, orhypothalamus tissue sample or in a body fluid (e.g., urine, CSF, orblood) in an amount lower than in a normal control sample; or (b) no orlower than normal amount of the IBC-1 receptor in a substantia nigra,pons, or hypothalamus tissue sample, would indicate that the patient issuffering from, or at risk for developing, a neural condition involvingdegeneration of substantia nigra, pons or hypothalamus cells. Such testscan be used on their own or, in conjunction with other procedures totest for invasive and metastatic breast cancer or degenerative neuraldiseases in appropriate subjects, e.g., human breast cancer patients, orpatients suspected of suffering from, or being at risk for developing, aneural condition involving degeneration of substantia nigra, pons orhypothalamus cells. These patients have symptoms of breast cancer,Parkinson's disease, or other neurological conditions. All humanspecimens (e.g., primary breast tumors from biopsies or surgicallyremoved tumors, and brain samples from autopsies) can be collected usingInstitutional Review Board approved protocols, snapped frozen on dryice, and stored at −80° C.

[0039] Methods of measuring mRNA levels in test cells or body fluids areknown in the art. In order to measure mRNA levels, cells in test samplescan be lysed and the levels of IBC-1 mRNA in the lysates or in RNApurified or semi-purified from the lysates determined by any of avariety of methods familiar to those in the art. Such methods include,without limitation, hybridization assays using detectably labeledIBC-1-specific DNA or RNA probes and quantitative or semi-quantitativeRT-PCR methodologies using appropriate IBC-1 gene-specificoligonucleotide primers. Alternatively, quantitative orsemi-quantitative in situ hybridization assays can be carried out using,for example, tissue sections or unlysed cell suspensions, and detectably(e.g., fluorescently or enzyme-) labeled DNA or RNA probes. Additionalmethods for quantifying MRNA include the RNA protection assay (RPA),cDNA and oligonucleotide microarrays, representation difference analysis(RDA), differential display, EST sequence analysis, and SAGE.

[0040] Methods of measuring protein levels in test cells or body fluidsare also known in the art. Many such methods employ antibodies (e.g.,monoclonal or polyclonal antibodies) that bind specifically to the IBC-1protein. In such assays, the antibody itself or a secondary antibodythat binds to it can be detectably labeled. Alternatively, the antibodycan be conjugated with biotin, and detectably labeled avidin (apolypeptide that binds to biotin) can be used to detect the presence ofthe biotinylated antibody. Combinations of these approaches (including“multi-layer sandwich” assays) familiar to those in the art can be usedto enhance the sensitivity of the methodologies. Some of theseprotein-measuring assays (e.g., ELISA or Western blot) can be applied tobodily fluids or to lysates of test cells, and others (e.g.,immunohistological methods or fluorescence flow cytometry) applied tohistological sections or unlysed cell suspensions. Methods of measuringthe amount of label will be depend on the nature of the label and areknown in the art. Appropriate labels include, without limitation,radionuclides (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P), enzymes (e.g.,alkaline phosphatase, horseradish peroxidase, luciferase, orβ-glactosidase), fluorescent moieties or proteins (e.g., fluorescein,rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g.,Qdot™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.). Other applicable assays include quantitativeimmunoprecipitation or complement fixation assays.

[0041] Amplification of a gene locus can be detected by a variety ofmethods known in the art. For example, the copy number of a gene locuscan be determined and compared by PCR amplification of genomic DNAprepared from a test sample and a control sample. Amplification of agene locus can also be identified by Southern blot analysis.Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location and an amount of the DNA sequence present in thechromosome.

[0042] The presence of an IBC-1 receptor on the surface of a test cellcan be determined by measuring the amount of IBC-1 bound to the cell.Methods of measuring ligand-receptor binding in test cells are alsoknown in the art. Many such methods involve contacting a ligand with areceptor (e.g., a receptor expressed on the surface of a cell), allowinga complex to form between the ligand and the receptor, and detecting thebound ligand as described above. The ligand of this invention can be theIBC-1 protein itself or a receptor-binding portion of the IBC-1 protein.As used herein, “a receptor-binding portion” of the IBC-1 protein is afragment of the protein that is shorter (e.g., having 10, 20, 30, 40,50, 60, 70, 80, or 90 consecutive amino acids of IBC-1) than thefull-length protein and has at least 5% (e.g., 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or more) of the ability of thefull-length protein to bind its receptor (e.g., as measured in acompetition assay). Fragments of interest can be made by recombinant,synthetic, or proteolytic digestive methods. Such fragments can then beisolated and tested for their ability to bind an IBC-1 receptor.

[0043] Generally, the level of IBC-1 or its receptor in diseased sampleswill be at least 2-fold (e.g., at least 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 10-fold, 15-fold, 20-fold, 40-fold, 60-fold, 80-fold,100-fold, 500-fold, 1,000-fold, or higher-fold) different (i.e., higherin samples from patients with invasive breast cancer, and lower insamples from patients with neural conditions involving degeneration ofsubstantia nigra, pons or hypothalamus cells) from that in the normalcounterpart samples.

[0044] Screening Assay

[0045] This invention provides methods (also referred to herein as“screening assays”) for identifying test compounds (e.g., proteins,peptides, peptidomimetics, peptoids, antibodies, small molecules orother drugs) that block the binding of IBC-1 to its receptor. Compoundsthus identified can be used to treat conditions characterized byover-activity of IBC-1 or its receptor, e.g., invasive breast cancer.

[0046] The test compounds of the present invention can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art. Such libraries include: peptide libraries, peptoid libraries(libraries of molecules having the functionalities of peptides, but witha novel, non-peptide backbone that is resistant to enzymaticdegradation; see, e.g., Zuckernann et al. (1994) J Med Chem 37:2678-85);spatially addressable parallel solid phase or solution phase libraries;synthetic libraries obtained by deconvolution or affinity chromatographyselection; and the “one-bead one-compound” libraries. Compounds in thelast three libraries can be peptides, non-peptide oligomers or smallmolecules (Lam (1997) Anticancer Drug Des 12:145). The test compoundscan also be antibodies generated against IBC-1 fragments identified ascritical for the binding of IBC-1 to its receptor by molecular modelingor mutational analysis.

[0047] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example, in: DeWitt et al. (1993) PNAS USA90:6909; Erb et al. (1994) PNAS USA 91:11422; Zuckermann et al. (1994) JMed Chem 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al.(1994) Angew Chem Int Ed Engl 33:2059; Carell et al. (1994) Angew ChemInt Ed Engl 33:2061; and Gallop et al. (1994) J Med Chem 37:1233.

[0048] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No.5,223,409), plasmids (Cull et al. (1992) PNAS USA 89:1865-1869), orphages (Scott and Smith (1990) Science 249:386-390; Devlin (1990)Science 249:404-406; Cwirla et al. (1990) PNAS USA 87:6378-6382; Felici(1991) J Mol Biol 222:301-310; and Ladner supra.).

[0049] To identify compounds that block the interaction between IBC-1and its receptor, a reaction mixture containing IBC-1 (or areceptor-binding portion of it) and a cell (or a cell membrane fractionor cell ghost or lipid vesicle) expressing the IBC-1 receptor isprepared under conditions and for a time sufficient to allow the tworeagents to form a complex. Cells expressing the IBC-1 receptor includecertain breast cancer cells (e.g., an invasive breast cancer cell), andcertain neural cells (e.g., a substantia nigra, pons or hypothalamuscell). Such cells can be easily identified by the techniques describedherein, or by determining whether labeled IBC-1 binds to the cell. Ithas been found that cells that express IBC-1 also express the IBC-1receptor. Alternatively, one can prepare such a cell by expressing arecombinant form of the receptor in a cultured cell.

[0050] The test compound can be initially included in the reactionmixture, or can be added at a time subsequent to the addition of IBC-1and its receptor. Control reaction mixtures are incubated without thetest compound. The formation of a complex between IBC-1 and its receptoris then detected, e.g., by detecting IBC-1 bound to its receptor asdescribed above. The formation of a complex in the control reaction, butnot in the reaction mixture containing the test compound, indicates thatthe compound blocks the interaction of IBC-1 and its receptor.

[0051] Generally, a test compound whose presence reduces IBC-1 bindingto its receptor at least 1.5 fold (e.g., at least 2-fold, 4-fold,6-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, or 100,000-fold canbe useful as a cancer therapeutic agent. Two types of IBC-1 receptorshave been identified: one with low affinity and one with high affinity(FIG. 2C). A cell expressing a low-affinity IBC-1 receptor can be usedto identify compounds that block binding of IBC-1 to the low-affinityreceptor; a cell expressing a high-affinity IBC-1 receptor can be usedto identify compounds that block binding of IBC-1 to the high-affinityreceptor. A compound that blocks IBC-1 binding to a high-affinityreceptor is more likely to be a cancer therapeutic agent.

[0052] Methods of Treating Cancer and Degenerative Neural Diseases

[0053] This invention provides methods for treating or preventinginvasive and metastatic breast cancer. “Prevention” should mean thatsymptoms of the disease (e.g., invasive and metastatic cancer) areessentially absent. Patients to be treated can be identified, forexample, by determining the IBC-1 mRNA, IBC-1 protein, or genomic IBC-1DNA level in a test sample prepared from a patient. If a patient hasbreast cancer and the IBC-1 mRNA or IBC-1 protein is present in a breastcancer tissue sample or a body fluid at a level higher than that in acontrol sample, or the IBC-1 gene is amplified in a breast cancer tissuesample, the patient is a candidate for treatment with an effectiveamount of compound that blocks binding of IBC-1 to its receptor.

[0054] This invention also provides methods for treating patientssuffering from, or at risk for developing, degenerative neuralconditions. If the IBC-1 MRNA or IBC-1 protein is present in a neuraltissue sample (e.g., a substantia nigra, pons or hypothalamus tissuesample) or a body fluid from a patient at a level lower than normal, thepatient is treated by administering IBC-1 or its agonist to the patientsuch that the IBC-1 or agonist reaches the affected tissue in the brain,in an effective amount to delay, prevent, or reverse neuraldegeneration.

[0055] The level of IBC-1 mRNA, IBC-1 proteins, or genomic IBC-1 DNA ina test sample can be determined by methods described above, or any othermethods known in the art.

[0056] The treatment methods can be performed in vivo or ex vivo, aloneor in conjunction with other drugs and/or radiotherapy.

[0057] (1) Methods of Treating or Preventing Invasive and MetastaticBreast Cancer

[0058] In one in vivo approach, a therapeutic compound (e.g., a compoundthat blocks the binding of IBC-1 to its receptor) itself is administeredto the subject. As used herein, a “therapeutic compound” can mean acompound the administration of which results in complete abolishment ofthe symptoms of a disease or a decrease in the severity of the symptomsof the disease. Generally, the compound will be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally or by intravenous (i.v.) infusion, or injected orimplanted subcutaneously, intramuscularly, intrathecally,intraperitoneally, intrarectally, intravaginally, intranasally,intragastrically, intratracheally, or intrapulmonarily. For treatment ofinvasive and metastatic breast cancer, the compound is preferablydelivered directly to tumor cells, e.g., to a tumor or a tumor bedfollowing surgical excision of the tumor, in order to kill any remainingtumor cells. For protection of breast cancer invasion and metastases,the compound can be administered (by any of the above routes) to, forexample, a patient that has not yet developed detectable invasion andmetastases but whose primary tumor was found to express IBC-1. Thedosage required depends on the choice of the route of administration;the nature of the formulation; the nature of the patient's illness; thesubject's size, weight, surface area, age, and sex; other drugs beingadministered; and the judgment of the attending physician. Suitabledosages are in the range of 0.01-100.0 mg/kg. Wide variations in theneeded dosage are to be expected in view of the variety of compoundsavailable and the different efficiencies of various routes ofadministration. For example, oral administration would be expected torequire higher dosages than administration by i.v. injection. Variationsin these dosage levels can be adjusted using standard empirical routinesfor optimization as is well understood in the art. Administrations canbe single or multiple (e.g., 2-fold, 3-fold, 4-fold, 6-fold, 8-fold,10-fold, 20-fold, 50-fold, 100-fold, 150-fold, or more fold).Encapsulation of the compound in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

[0059] Therapeutic compounds useful for treating or preventingmetastatic breast cancer include, but are not limited to, antagonisticfragments of IBC-1, antibodies specific for IBC-1 (e.g., any of theIBC-1-specific antibodies disclosed herein), and/or antibodies specificfor IBC-1 receptor. These antibodies would of course have to be screenedfor antagonistic activity and lack of agonistic activity.

[0060] Alternatively, a polynucleotide containing a nucleic acidsequence that is transcribed into an anti-sense RNA complementary toIBC-1 mRNA (the full-length mRNA sequence or a suitable portion thereof)can be delivered to breast cancer cells. Polynucleotides can bedelivered to breast cancer cells by, for example, the use of polymeric,biodegradable microparticle or microcapsule devices known in the art.Another way to achieve uptake of the nucleic acid is using liposomes,prepared by standard methods. The vectors can be incorporated alone intothese delivery vehicles or co-incorporated with tissue-specific ortumor-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano etal. (1995) J Mol Med 73:479). Tissue specific targeting can be achievedby the use of tissue-specific transcriptional regulatory elements (TRE)which are known in the art. Delivery of “naked DNA” (i.e., without adelivery vehicle) to an intramuscular, intradermal, or subcutaneous siteis another means to achieve in vivo expression.

[0061] The polynucleotide can include one or more sequencescomplementary to the sense strand of IBC-1 DNA and a catalytic sequenceknown to be responsible for mRNA cleavage (see, e.g., U.S. Pat. No.5,093,246 and Haselhoff and Gerlach (1988) Nature 334:585-591). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in an IBC-1 mRNA. See, e.g., U.S.Pat. Nos. 4,987,071 and 5,116,742. Alternatively, an IBC-1 mRNA can beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel and Szostak (1993)Science 261:1411-1418.

[0062] In the relevant polynucleotides (e.g., expression vectors), thenucleic acid sequence encoding the anti-sense RNA is operatively linkedto a promoter or enhancer-promoter combination. Enhancers provideexpression specificity in terms of time, location, and level. Unlike apromoter, an enhancer can function when located at variable distancesfrom the transcription initiation site, provided a promoter is present.An enhancer can also be located downstream of the transcriptioninitiation site.

[0063] Suitable expression vectors include plasmids and viral vectorssuch as herpes viruses, retroviruses, vaccinia viruses, attenuatedvaccinia viruses, canary pox viruses, adenoviruses and adeno-associatedviruses, among others.

[0064] Double-stranded interfering RNA (RNAi) homologous to IBC-1 DNAcan also be used to reduce the production of IBC-1. See, e.g., Fire etal. (1998) Nature 391:806-811, Romano and Masino (1992) Mol. Microbiol.6:3343-3353, Cogoni et al. EMBO J. 15:3153-3163, Cogoni and Masino(1999) Nature 399:166-169, Misquitta and Paterson (1999) Proc. Natl.Acad. Sci. USA 96:1451-1456, and Kennerdell and Carthew (1998) Cell95:1017-1026.

[0065] The sense and anti-sense RNA strands can be individuallyconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, each strand can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecule or to increase the physical stability of theduplex formed between the sense and anti-sense strands, e.g.,phosphorothioate derivatives and acridine substituted nucleotides. Thesense or anti-sense strand can also be produced biologically using anexpression vector into which a target IBC-1 sequence (full-length or afragment) has been subcloned in a sense or anti-sense orientation. Thesense and anti-sense RNA strands can be annealed in vitro beforedelivery of the dsRNA to breast cancer cells. Alternatively, annealingcan occur in vivo after the sense and anti-sense strands aresequentially delivered to the cancer cells.

[0066] Double-stranded RNA interference can also be achieved byintroducing into breast cancer cells a polynucleotide from which senseand anti-sense RNAs can be transcribed under the direction of separatepromoters, or a single RNA molecule containing both sense and anti-sensesequences can be transcribed under the direction of a single promoter.

[0067] Polynucleotides can be administered in a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers arebiologically compatible vehicles that are suitable for administration toa human, e.g., physiological saline or liposomes. A therapeuticallyeffective amount is an amount of the polynucleotide that is capable ofproducing a medically desirable result (e.g., decreased IBC-1expression) in a treated patient. As is well known in the medical arts,the dosage for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages will vary, but apreferred dosage for administration of polynucleotide is fromapproximately 10⁶ to 10¹² copies of the polynucleotide molecule. Thisdose can be repeatedly administered, as needed. Routes of administrationcan be any of those listed above.

[0068] (2) Methods of Treating Degenerative Neural Conditions

[0069] A. In vivo Approaches

[0070] An IBC-1 or an IBC-1 agonist can be administered to a patient whohas, or is likely to develop, a degenerative neural condition.Generally, IBC-1 or an IBC-1 agonist (e.g., an agonistic IBC-1 receptorantibody) will be suspended in a pharmaceutically-acceptable carrier(e.g., physiological saline) and administered orally or by inhalation ori.v. infusion, or injected or implanted subcutaneously, intramuscularly,intrathecally, intraperitoneally, intrarectally, intravaginally,intranasally, intragastrically, intratracheally, or intrapulmonarily.For treatment of degenerative neural conditions, IBC-1, as well as thoseIBC-1 agonists small enough to cross the blood-brain barrier, does nothave to be delivered directly to neural cells, although injection of thedrug or implantation of a drug-releasing barrier device or cells in thebrain are options. The dosage required depends on the choice of theroute of administration; the nature of the formulation; the nature ofthe patient's illness; the subject's size, weight, surface area, age,and sex; other drugs being administered; and the judgment of theattending physician. Suitable dosages are in the range of 0.01-100.0mg/kg. Wide variations in the needed dosage are to be expected in viewof the variety of compounds available and the different efficiencies ofvarious routes of administration. For example, oral administration wouldbe expected to require higher dosages than administration by i.v.injection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Administrations can be single or multiple (e.g., 2-fold,3-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold,150-fold, or more fold). Encapsulation of the compound in a suitabledelivery vehicle (e.g., polymeric microparticles or implantable devices)may increase the efficiency of delivery, particularly for oral delivery.

[0071] B. Ex vivo Approaches

[0072] An ex vivo strategy for treating patients with degenerativeneural conditions can involve transfecting or transducing cells obtainedfrom the subject with a polynucleotide encoding IBC-1, pro-IBC-1, or anIBC-1 agonist. Alternatively, a cell can be transfected in vitro with avector designed to insert, by homologous recombination, a new, activepromoter upstream of the transcription start site of the naturallyoccurring endogenous IBC-1 gene in the cell's genome. Such methods,which “switch on” an otherwise largely silent gene, are well known inthe art. After selection and expansion of a cell that expresses IBC-1 ata desired level, the transfected or transduced cells are then returnedto the subject. The cells can be any of a wide range of types including,without limitation, hemopoietic cells (e.g., bone marrow cells,macrophages, monocytes, dendritic cells, T cells, or B cells),fibroblasts, epithelial cells, endothelial cells, keratinocytes, ormuscle cells. Such cells act as a source of secreted IBC-1 or an IBC-1agonist for as long as they survive in the subject.

[0073] The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of IBC-1 or an IBC-1 agonist. These methods are known in theart of molecular biology. The transduction step is accomplished by anystandard means used for ex vivo gene therapy, including calciumphosphate, lipofection, electroporation, viral infection, and biolisticgene transfer. Alternatively, liposomes or polymeric microparticles canbe used. Cells that have been successfully transduced can then beselected, for example, for expression of IBC-1 or an IBC-1 agonist. Thecells may then be injected or implanted into the patient.

[0074] The following examples are meant to illustrate, not limit, theinvention.

EXAMPLES

[0075] Materials and Methods

[0076] (1) Cell Lines and Tissue Specimens

[0077] Breast cancer cell lines were obtained from American Type CultureCollection (Manassas, Va.), or were generously provided by Drs. SteveEthier (University of Michigan), Gail Tomlinson (University of Texas),and Arthur Pardee (Dana-Farber Cancer Institute). Cells were grown inmedia recommended by the providers. Primary breast tumor samples wereobtained from Brigham and Women's Hospital, Massachusetts GeneralHospital, University Hospital Zagreb (Zagreb, Croatia), or DukeUniversity Medical Center. Immediately after removal from the patients,the samples were snap frozen on dry ice, and stored at −80° C. Brainsamples were collected from autopsies performed at Brigham and Women'sHospital or Duke University Medical Center. All human specimens werecollected using Institutional Review Board approved protocols, and allpatient identifiers were removed prior to being transported to thelaboratory.

[0078] (2) RNA Preparation, MRNA in situ Hybridization, and NorthernBlot Analysis

[0079] RNA isolation, RT-PCR and Northern blot analyses were performedas described (Polyak et al. (1997) Nature 389:300-305). Human multipletissue Northern blots were purchased from Clontech (Palo Alto, Calif.).mRNA in situ hybridization using paraffin sections and digitonin-labeledriboprobes was performed following a protocol developed by St. Croix etal. (2000) Science 289:1197-1202. Frozen sections were hybridizedfollowing a protocol obtained from Dr. Qiufu Ma (Dana-Farber CancerInstitute) with minor modifications (Qian et al. (2001) Genes Dev15:2533-2545).

[0080] (3) Expression of IBC-1 in Mammalian Cells and in Bacteria

[0081] To produce recombinant IBC-1 protein in large quantities inmammalian cells, a cDNA encoding human IBC-1 without the signal sequencewas generated by PCR. The PCR fragment was cloned into the pSGHVOvector; this resulted in a vector encoding human growth hormone (hGH) asan amino terminal fusion partner joined to IBC-1 by a linker containinga histidine affinity tag and a tobacco etch virus protease site (Leahyet al. (2000) Protein Expr Purif 20:500-506). For ligand binding assays,alkaline phosphatase (AP)-IBC-1 fusion proteins were generated using anAP-TAG-5 expression vector (GenHunter, Nashville, Tennessee). Mammaliancells were transfected with Fugene6™ (Roche, Indianapolis, Ind.) orLipofectamine™ (LifeTechnologies, Rockville, Md.) reagents.

[0082] For bacterial expression, the IBC-1 cDNA was PCR amplified andcloned into pQE-30 in frame with an N-terminal hexahistidine tag. Theconstruct was transformed into MJ15 [pREP4] bacteria (Qiagen, Valencia,Calif.), and the recombinant IBC-1 protein was purified using denaturingbuffer and Ni-NTA beads (Qiagen, Valencia, Calif.).

[0083] (4) Antibodies, Immunoblot Analyses, and in vitro Translation

[0084] A polyclonal anti-IBC-1 antibody was generated against asynthetic peptide RQAPKPRKQRSS (SEQ ID NO:7) corresponding to aminoacids 53-64 of pro-IBC-1 (Zymed, San Francisco, Calif.). Antibodiesagainst alkaline phosphatase and cachectic factor-1 were purchased fromGenHunter (Nashville, Tenn.) and Alpha Diagnostic (San Antonio, Tex.),respectively. Immnunoblot analyses were performed as described (Krop etal. (2001) PNAS USA 98:9796-9801). Coupled in vitro transcription andtranslation reactions were performed using a PCR-generated nucleic acidencoding an IBC-1 fragment containing a C-terminal hexahistidine tag, aT7 TNT kit (Promega, Madison, Wis.) and an ³⁵S-labelled Promix™ aminoacid mixture (Amersham, Piscataway, N.J.). Proteins were purified withNiNTA beads (Qiagen, Valencia, Calif.), and resolved on 16% Tris-tricinegels (Invitrogen, Carlsbad, Calif.).

[0085] (5) Ligand Binding Assays

[0086] In vivo and in vitro ligand binding assays with primary tissuesand cell lines using AP-IBC-1 were performed essentially as described(Flanagan and Leder (1990) Cell 63:185-194). Briefly, frozen sections ofvarious human tissue specimens were fixed, incubated with eitherAP-IBC-1 fusion protein or AP control conditioned medium, rinsed, andthen incubated with AP substrate, forming a blue/purple precipitate. Forin vitro assays, cells were incubated in suspension with conditionedmedium containing either AP alone or AP-IBC-1 fusion protein, rinsed,and then assayed for the activity of bound AP. For ligand competitionexperiments, G361 cells were pre-incubated with purified recombinantHis-HID-5/psoriasin (1.93 μM) or His-IBC-1 (0.567 μM) followed byaddition of AP or AP-IBC-1.

Example 1

[0087] Identification of an IBC-1 Gene Encoding the Human Proteolysisand Cachexia Inducing Factor

[0088] Analyses of SAGE libraries derived from normal mammary epithelialcells and in situ, invasive, and metastatic breast carcinomas identifieda SAGE tag present only in libraries generated from invasive andmetastatic breast carcinomas (Porter et al. (2001) Cancer Res61:5697-5702). This tag was absent in 96 other SAGE libraries generatedfrom various human normal and cancerous tissue types (Lal et al. (1999)Cancer Res 59:5403-5407). The gene corresponding to this SAGE tag wasnamed IBC-1.

[0089] Searching the human genome sequence with a 15 base-pair sequencecontaining the IBC-1 SAGE tag and adjacent NlaIII site (CATGACGTTAAAGAC;SEQ ID NO:8), the inventors identified a genomic clone containing thistag. Using the GenScan program, it was predicted that this genomicregion encodes a gene of 4 exons with the IBC-1 SAGE tag in the lastexon following the last NlaIII site (Burge and Karlin (1997) J Mol Biol268:78-94). Based on the predicted coding sequence, primers for the most5′ and 3′ ends of the cDNA were designed, and RT-PCR analysis of mRNAderived from the breast carcinomas used for SAGE was performed. Usingthis approach, a 400 bp fragment was obtained, thus confirming that theregion encodes a transcribed gene. To confirm that the sequence of this400 bp fragment matches that of the genomic clone, the fragment wassequenced. The sequence showed that there was a small, additionaltranscribed exon not identified by the GenScan program. Therefore, thecomplete IBC-1 gene contains 5 exons, and encodes a 110 amino acidprotein containing an N-terminal signal peptide (FIGS. 1A and 1B).

[0090] The IBC-1 cDNA sequence is as follows:    GAAGCATGAGGTTCATGACTCTCCTCTTCCTGACAGCTCTGGCAGGAGCCCTGGT (SEQ IDNO: 1) CTGTGCCTATGATCCAGAGGCCGCCTCTGCCCCAGGATCGGGGAACCCTTGCCATGAAGCATCAGCAGCTCAAAAGGAAAATGCAGGTGAAGACCCAGGGTTAGCCAGACAGGCACCAAAGCCAAGGAAGCAGAGATCCAGCCTTCTGGAAAAAGGCCTAGACGGAGCAAAAAAAGCTGTGGGGGGACTCGGAAAACTAGGAAAAGATGCAGTCGAAGATCTAGAAAGCGTGGGTAAAGGAGCCGTCCATGACGTTAAAGACGTCCTTGACTCAGTACTATAGCTGTAAGGAGAAGCTGAGAAATGATACCCAGGAGCAGCAGGCTTTACGTCTTCAGCCTAAAACCTA A

[0091] The IBC-1 cDNA (SEQ ID NO:1) is 405 nucleotides in length. Thenucleic acid sequence includes an initiation codon (ATG) and atermination codon (TAG) that are underlined above. The region betweenand inclusive of the initiation codon and the termination codon is amethionine-initiated coding sequence of 333 nucleotides including thetermination codon. This coding sequence is given SEQ ID NO:2. The codingsequence encodes a 110 amino acid protein (SEQ ID NO:3):    MRFMTLLFLTALAGALVCAYDPEAASAPGSGNPCHEASAAQKENAGEDPGLARQA (SEQ ID NO:3) PKPRKQRSSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL

[0092] The coding region of IBC-1 cDNA (SEQ ID NO:1) was found to beincluded in a previously identified nucleic acid sequence encoding ahuman cachexia-associated protein (HCAP; Akerblom et al., U.S. Pat. No.5,834,192). However, the two sequences differ by one nucleotide withinthe 3′-untranslated region: the base at position 388 of SEQ ID NO:1(shown above in bold) is a cytosine, whereas the hcap cDNA sequence hasa thymine at the corresponding position. In addition, the predictedIBC-1 protein has very limited homology to a lacritin protein and to atranslated EST derived from the cerebral cortex (FIG. 1C). Lacritin is asecretion-enhancing and growth-promoting factor recently identified fromhuman lacrimal gland (Sanghi et al. (2001) J Mol Biol 310:127-139). TheEST expressed in the cerebral cortex encodes an uncharacterized proteincontaining a repetitive sequence ETPA found in several secretedproteins, including sialidase and neurofilamine H.

[0093] Further lower stringency searches of the Unigene and Genbankdatabases using the predicted IBC-1 amino acid sequence revealed that aportion of IBC-1 nearly matches a 20 amino acid peptide derived from themouse PIF (Proteolysis Inducing Factor) or cachectic factor (CF), and anoverlapping portion exactly matches the sequence of a 30 amino acidputative neural survival-promoting peptide (Cariuk et al. (1997) Br JCancer 76:606-613; and Cunningham et al. (1998) J Neurosci18:7047-7060). These polypeptides have been characterized as havingbiological and biochemical activities, but the genes encoding them havenot been identified. The neural survival-promoting peptide wasidentified from the media of mouse HN33.1 hippocampal neurons and humanY79 retinoblasts treated with hydrogen peroxide, and subsequently shownto enhance neural survival following an oxidative insult. The cachecticand proteolysis inducing factor was identified as a 24-kDa glycoproteinproduced by the cachexia-inducing MAC 16 murine colon adenocarcinoma inmice, and later shown to be present in the urine of cachectic cancerpatients (McDevitt and Tisdale (1992) Br J Cancer 66:815-820; Todorov etal. (1996) Cancer Res 56:1256-1261; and Todorov et al. (1996) Nature379:739-742). In subsequent studies, the 24 kDa cachectic factor wasshown to induce muscle protein degradation both in vivo in mice and invitro in C2C12 mouse myoblasts (Todorov et al. (1996) Cancer Res56:1256-1261; and Smith et al. (1999) Cancer Res 59:5507-5513).

[0094] The IBC-1 cDNA is predicted to encode an ˜11 kDa protein, whichwas confirmed by in vitro translation reaction. The amino acid sequenceof the tryptic peptide obtained from the murine 24 kDa proteolysisinducing/cachectic factor (YDPEAASAPGSGNPSHEASA; SEQ ID NO:9) almostexactly matches amino acids 20-39 of the predicted IBC-1 sequence, butdoes not match to any other characterized or predicted proteins in theUnigene and Genbank databases. However, the amino acid sequence of IBC-1contains no predicted N-glycosylation sites, whereas the proteolysis andcachexia inducing protein was reported to be heavily glycosylated.

[0095] To determine if IBC-1 and the proteolysis inducing/cachecticfactor are antigenically related, an immunoblot analysis of variousIBC-1 fusion proteins purified from bacterial and mammalian cells wasperformed using a custom made anti-IBC-1 peptide antibody and acommercially available anti-cachectic factor peptide antibody. Theseanalyses confirmed that IBC-1 and the proteolysis inducing/cachecticfactor are likely to be identical or at least antigenically related, butthe reason for the difference between the reported natural (˜24 kDa) andthe recombinant (˜11-13 kDa) protein sizes is unclear. One possibilityis that the anti-IBC-1 antibody used in the present studies and thecommercial anti-CF peptide antibodies do not recognize the glycosylatedor dimerized form of the proteolysis inducing/cachectic protein. Inaddition, the inventors have not been able to express the IBC-1 proteinat detectable levels in most cell types, so it may be translated in acell type-specific manner or it may be very unstable.

Example 2

[0096] Expression Pattern of IBC-1 in Normal and Cancerous Tissues

[0097] Northern blot analyses of multiple breast tumors using an IBC-1cDNA probe identified a single -400 bp hybridizing mRNA, indicating thatthe 400 bp cDNA fragment described above corresponds to the full-lengthtranscript. Northern blot, RT-PCR, and mRNA in situ hybridizationanalyses of normal breast organoids (freshly isolated mammary ducts),primary breast carcinomas, and breast cancer cell lines demonstratedthat IBC-1 is not expressed in normal mammary epithelium nor in themajority of breast cancer cell lines and tumors. Interestingly, all thebreast tumors (6 out of 55 total) that showed high IBC-1 expressionlevels were poorly differentiated (grade III) and stronglyerbB2-expressing tumors, and five out of the six IBC-1 positive tumorshad multiple metastatic lymph nodes. However, due to the relativelysmall sample size, among these tumor characteristics, only theoverexpression of erbB2 showed a nearly significant (Fisher exact testP=0.06) association with IBC-1 expression. These data indicate thatIBC-1 expression is not a common event in breast carcinomas, but definesa particularly aggressive tumor phenotype.

[0098] Analyses of 100 SAGE libraries derived from multiple normal andcancerous human tissues and cell lines suggested that IBC-1 is expressedonly in a subset of breast carcinomas. To further investigate IBC-1expression, the IBC-1 cDNA was hybridized against a tissue expressionarray panel containing mRNA from 76 normal human adult and fetal tissuetypes. IBC-1 was found to be expressed only in two regions of the brain:in the pons, and at a lower level, in the paracentral gyrus of thecerebral cortex. This restricted expression pattern suggests that IBC-1would be useful as a breast cancer diagnostic or prognostic marker. Inaddition, these observations imply that IBC-1-expressing tumors may haveacquired a neuronal phenotype. In order to test the latter hypothesis,the expression of several neural markers (e.g., chromogranin A,synaptogenin, neuronal enolase) in the breast tumor samples was analyzedby Northern blot or SAGE analysis, but found no correlation between theexpression of these genes and that of IBC-1.

[0099] The SAGE libraries from which IBC-1 was identified were generatedfrom unpurified invasive and metastatic breast carcinomas containingstromal fibroblasts, lymphocytes, endothelial cells, and other celltypes. To characterize the expression of IBC-1 at the cellular level,mRNA in situ hybridization was performed on sections of two tumors knownto express IBC-1 based on Northern blot analysis and ten additionaltumors that were similar to them based on expression profiling andclustering analysis. Four out of the latter ten tumors contained someIBC-1 positive tumor cells, further suggesting that IBC-1 expressiondefines a biologically relevant subset of breast carcinomas. Intense redor black (depending on the MRNA in situ hybridization method used)staining in the anti-sense slide demonstrates that IBC-1 is expressed intumor cells, but not in stromal fibroblasts, endothelial cells, orlymphoid cells. No signal was observed in adjacent normal mammaryepithelial cells. Interestingly, in some samples, all tumor cells werestrongly IBC-1 positive, while in others, only a subset of the tumorcells showed high IBC-1 expression, indicating that IBC-1 is useful inidentifying intra-tumoral clonal heterogeneity.

[0100] To analyze the expression of IBC-1 at the protein level, a rabbitpolyclonal anti-IBC-1 antibody was generated using a synthetic IBC-1peptide as an immunogen and affinity-purified. To determine whether theexpression of IBC-1 correlates with histo-pathologic or clinicalcharacteristics of breast tumors, an immunohistochemical analysis of 722breast tumors collected from 8 different cohorts was performed using thepolyclonal anti-IBC-1 antibody. Overall, only 6-11% (depending on thepatient cohort) of these tumors were IBC-1 positive. Statisticalanalysis of the immunohistochemistry data indicated that the expressionof IBC-1 was not significantly different between (a) in situ and primaryinvasive tumors and (b) distant metastases, although the number of insitu tumors and distant metastases were relatively low and only 1 ductalcarcinoma in situ (DCIS) was found to be IBC-1 positive. Similarly, noassociation was found between (a) IBC-1 expression and (b) estrogen andprogesterone receptor status, tumor size, the number of positive lymphnodes and the age of the patient. However, the expression of IBC-1correlated positively with erbB2 expression in a subset of the tumors.importantly, based on a logistic regression model, IBC-1 positiveprimary tumors were more likely to be stage 2 and 3 than stage 1 (LRtest, p-value=0.007), suggesting a role for IBC-1 in tumor progression.Finally, patients with IBC-1 positive tumors were somewhat more likelyto have a shorter disease free survival (calculated as time untildistant metastasis) and overall survival than patients with IBC-1negative tumors. Although this association did not reach statisticalsignificance (p-value=0.8 for overall survival and p-value=0.43 fordisease free survival) due to the relatively low number of IBC-1positive tumors with clinical data, it suggests that IBC-1 expressioncan be an independent indicator of poor prognosis.

Example 3

[0101] Identification of Putative Cell Surface IBC-1 Binding Protein(s)

[0102] Both cachectic factor and the neural survival peptide wereidentified as secreted proteins. Consistent with that, the IBC-1 cDNAencodes a 110 amino acid protein with a predicted 19 amino acidsecretory signal peptide. These data indicate that IBC-1 is likely toexecute its function through binding to a cell surface receptor. Todetermine if there is an IBC-1-binding cell surface protein(s), analkaline phosphatase-IBC-1 (AP-IBC-1) fusion protein to be used as aligand in receptor binding assays was generated (Flanagan and Leder(1990) Cell 63:185-194; FIG. 2A). Conditioned medium of AP-IBC-1 orcontrol AP expressing cells was used as an affinity reagent, much likean antibody, to stain normal and cancerous mammary tissue sections.Intense purple staining indicates the presence of an IBC-1-bindingprotein in invasive breast carcinoma with high endogenous IBC-1expression, but not in normal mammary epithelial and stromal cells.Interestingly, tumors that express IBC-1 demonstrated much more intensestaining than did tumors with low or undetectable endogenous IBC-1expression. These results indicate the presence of a cell surfaceIBC-1-binding protein in cancerous but not normal mammary epithelialcells in vivo, and indicate an autocrine mechanism of IBC-1 action.

[0103] Because IBC-1 is expressed in neurons of the pons, hypothalamusand midbrain, and because of IBC-1's role in cachexia, these tissueswere tested for their ability to bind IBC-1. Surprisingly, weak IBC-1binding to almost all neurons was seen. The most intense alkalinephosphatase staining (i.e., the strongest IBC-1 binding) was detected inneurons of the locus ceruleus, nucleus raphe pontis, substantia nigra,and the lateral hypothalamic nuclei, also known as the lateralhypothalamic area or zone.

[0104] To further test the binding characteristics of AP-IBC-1, in vitroligand binding assays were performed on various cell lines (FIG. 2B).Low level AP-IBC-1 binding was detected in all cell lines tested, andstronger binding was observed in human 21NT breast cancer, G361melanoma, mouse C2C12 myoblast, and CATH.a catecholaminergic neurons.The IBC-1 binding activity was completely abolished by pretreatment ofcells with trypsin, indicating that the IBC-1 binding activity is likelydue to the presence of a cell surface protein. Addition of purifiedrecombinant IBC-1, but not HID-5/psoriasin, significantly reducedAP-IBC-1 binding to G361 cells, indicating that the binding isIBC-1-specific. 21iNT and G361 cells express IBC-1; IBC-1 was shown toinduce proteolysis in C2C12 (Smith et al. (1999) Cancer Res59:5507-5513; and Todorov et al. (1999) Br J Cancer 80:1734-1737).CATH.a cells were derived from a brain tumor of transgenic miceexpressing the SV40 T antigen under the control of the rat tyrosinehydroxylase gene promoter (Suri et al. (1993) J Neurosci 13:1280-1291).CATH.a cells have catecholaminergic neuronal phenotype, and are highlysensitive to apoptosis induced by dopamine and hydrogen-peroxide(Masserano et al.(1996) Mol Pharmacol 50:1309-1315). Although the amountof bound AP-IBC-1 may be influenced by cell size, the difference in thesize of these cells is unlikely to account entirely for the observeddifferences in AP-IBC-1 binding. Therefore, cells that respond to IBC-1are likely to express an increased number of or higher affinity IBC-1binding proteins.

[0105] To further characterize the AP-IBC-1-putative IBC-1 receptorinteraction, more detailed binding assays were performed on G361melanoma cells (FIG. 2C). Scatchard plot analysis shows two bindingslopes: one with a moderately high affinity (Kd=37.5 nM, 1.7×10⁴ bindingsites/cell) and another with much lower affinity (Kd=360 nM, 3.5×10⁵binding sites/cell).

Example 4

[0106] Amplification of IBC-1 Gene in Breast Tumors

[0107] The IBC-1 gene is localized to chromosome 12q13, an areapreviously implicated in various malignancies. Intriguingly, based onour SAGE data, two genes nearest to IBC-1 (LACRT and PPPlRlA) were alsohighly and specifically expressed in the same tumor samples that highlyexpressed IBC-1. This led us to hypothesize that the overexpression ofIBC-1 in these breast tumors may be due to genetic amplification. Inorder to test this hypothesis, a bacterial artificial chromosome (BAC)containing the IBC-1 gene was isolated and used for FISH (fluorescent insitu hybridization) analysis of normal breast tissue and four breasttumors that overexpressed IBC-1 (as determined by SAGE or Northernblot/mRNA in situ hybridization). The analysis showed that there is amoderate- to high-level gain of the IBC-1 genomic locus in tumors thatoverexpress IBC-1. Since several known oncogenes, including CDK4, SAS,GLI, and MDM2, are also localized to chromosome 12q13-15, FISH analysesusing BACs corresponding to these genes was carried out to determine ifthey are co-amplified with IBC-1. MDM2 and GLI1 were not amplified inthese tumors, while in a subset of the cells, both CDK4 and IBC-1 wereamplified. However, in tumors that overexpressed IBC-1, there was noevidence of CDK4 overexpression. These data show that a subset ofinvasive breast tumors overexpress and amplify IBC-1.

Example 5

[0108] Polyclonal IBC-specific Antibodies

[0109] As a custom antibody service ordered by the inventors, ZymedLaboratories, Inc., South San Francisco, Calif., produced twoIBC-1-specific rabbit polyclonal antibody preparations by immunizing(using standard procedures) rabbits with three different syntheticpeptides. One group of rabbits was immunized with a N-terminal IBC-1synthetic peptide fragment consisting of amino acids 45-64 of pro-IBC-1(AGEDPGLARQAPKPRKQRSS; SEQ ID NO:12), a second group of rabbits wasimmunized with a N-terminal IBC-1 synthetic peptide fragment consistingof amino acids 53-64 of pro-IBC-1 (RQAPKPRKQRSS; SEQ ID NO:7), and athird group of rabbits was immunized with a C-terminal IBC-1 syntheticpeptide fragment consisting of amino acids 86-103 of pro-IBC-1(DAVEDLESVGKGAVHDVK; SEQ ID NO:13). Antibodies specific for theC-terminus of IBC-1 are especially useful in that the C-terminus ofIBC-1 is more stable than the N-terminus and thus antibodies specificfor the C-terminus provide a particularly sensitive means for testingfor the expression of, or the presence of, IBC-1 protein in, forexample, test cells (e.g., breast epithelial cells), tissues, or bodilyfluids (e.g., blood, urine, or sweat) of interest. The inventors found,by western blot analysis, that the three polyclonal antibodies producedby immunizing rabbits with the above-described three IBC-1 syntheticpeptide fragments bound to: recombinant IBC-1 produced in bacteria;IBC-1 expressed in breast cancer cells; and IBC-1 present in sweat (datanot shown).

OTHER EMBODIMENTS

[0110] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1 13 1 405 DNA Homo sapiens CDS (6)...(335) 1 gaagc atg agg ttc atg actctc ctc ttc ctg aca gct ctg gca gga gcc 50 Met Arg Phe Met Thr Leu LeuPhe Leu Thr Ala Leu Ala Gly Ala 1 5 10 15 ctg gtc tgt gcc tat gat ccagag gcc gcc tct gcc cca gga tcg ggg 98 Leu Val Cys Ala Tyr Asp Pro GluAla Ala Ser Ala Pro Gly Ser Gly 20 25 30 aac cct tgc cat gaa gca tca gcagct caa aag gaa aat gca ggt gaa 146 Asn Pro Cys His Glu Ala Ser Ala AlaGln Lys Glu Asn Ala Gly Glu 35 40 45 gac cca ggg tta gcc aga cag gca ccaaag cca agg aag cag aga tcc 194 Asp Pro Gly Leu Ala Arg Gln Ala Pro LysPro Arg Lys Gln Arg Ser 50 55 60 agc ctt ctg gaa aaa ggc cta gac gga gcaaaa aaa gct gtg ggg gga 242 Ser Leu Leu Glu Lys Gly Leu Asp Gly Ala LysLys Ala Val Gly Gly 65 70 75 ctc gga aaa cta gga aaa gat gca gtc gaa gatcta gaa agc gtg ggt 290 Leu Gly Lys Leu Gly Lys Asp Ala Val Glu Asp LeuGlu Ser Val Gly 80 85 90 95 aaa gga gcc gtc cat gac gtt aaa gac gtc cttgac tca gta cta 335 Lys Gly Ala Val His Asp Val Lys Asp Val Leu Asp SerVal Leu 100 105 110 tagctgtaag gagaagctga gaaatgatac ccaggagcagcaggctttac gtcttcagcc 395 taaaacctaa 405 2 333 DNA Homo sapiens 2atgaggttca tgactctcct cttcctgaca gctctggcag gagccctggt ctgtgcctat 60gatccagagg ccgcctctgc cccaggatcg gggaaccctt gccatgaagc atcagcagct 120caaaaggaaa atgcaggtga agacccaggg ttagccagac aggcaccaaa gccaaggaag 180cagagatcca gccttctgga aaaaggccta gacggagcaa aaaaagctgt ggggggactc 240ggaaaactag gaaaagatgc agtcgaagat ctagaaagcg tgggtaaagg agccgtccat 300gacgttaaag acgtccttga ctcagtacta tag 333 3 110 PRT Homo sapiens 3 MetArg Phe Met Thr Leu Leu Phe Leu Thr Ala Leu Ala Gly Ala Leu 1 5 10 15Val Cys Ala Tyr Asp Pro Glu Ala Ala Ser Ala Pro Gly Ser Gly Asn 20 25 30Pro Cys His Glu Ala Ser Ala Ala Gln Lys Glu Asn Ala Gly Glu Asp 35 40 45Pro Gly Leu Ala Arg Gln Ala Pro Lys Pro Arg Lys Gln Arg Ser Ser 50 55 60Leu Leu Glu Lys Gly Leu Asp Gly Ala Lys Lys Ala Val Gly Gly Leu 65 70 7580 Gly Lys Leu Gly Lys Asp Ala Val Glu Asp Leu Glu Ser Val Gly Lys 85 9095 Gly Ala Val His Asp Val Lys Asp Val Leu Asp Ser Val Leu 100 105 110 491 PRT Homo sapiens 4 Tyr Asp Pro Glu Ala Ala Ser Ala Pro Gly Ser GlyAsn Pro Cys His 1 5 10 15 Glu Ala Ser Ala Ala Gln Lys Glu Asn Ala GlyGlu Asp Pro Gly Leu 20 25 30 Ala Arg Gln Ala Pro Lys Pro Arg Lys Gln ArgSer Ser Leu Leu Glu 35 40 45 Lys Gly Leu Asp Gly Ala Lys Lys Ala Val GlyGly Leu Gly Lys Leu 50 55 60 Gly Lys Asp Ala Val Glu Asp Leu Glu Ser ValGly Lys Gly Ala Val 65 70 75 80 His Asp Val Lys Asp Val Leu Asp Ser ValLeu 85 90 5 23 PRT Homo sapiens 5 Tyr Asp Pro Glu Ala Ala Ser Ala ProGly Ser Gly Asn Pro Cys His 1 5 10 15 Glu Ala Ser Ala Ala Gln Lys 20 622 PRT Homo sapiens 6 Glu Asn Ala Gly Glu Asp Pro Gly Leu Ala Arg GlnAla Pro Lys Pro 1 5 10 15 Arg Lys Gln Arg Ser Ser 20 7 12 PRT Homosapiens 7 Arg Gln Ala Pro Lys Pro Arg Lys Gln Arg Ser Ser 1 5 10 8 15DNA Homo sapiens 8 catgacgtta aagac 15 9 20 PRT Mus musculus 9 Tyr AspPro Glu Ala Ala Ser Ala Pro Gly Ser Gly Asn Pro Ser His 1 5 10 15 GluAla Ser Ala 20 10 138 PRT Homo sapiens 10 Met Lys Phe Thr Thr Leu LeuPhe Leu Ala Ala Val Ala Gly Ala Leu 1 5 10 15 Val Tyr Ala Glu Asp AlaSer Ser Asp Ser Thr Gly Ala Asp Pro Ala 20 25 30 Gln Glu Ala Gly Thr SerLys Pro Asn Glu Glu Ile Ser Gly Pro Ala 35 40 45 Glu Pro Ala Ser Pro ProGlu Thr Thr Thr Thr Ala Gln Glu Thr Ser 50 55 60 Ala Ala Ala Val Gln GlyThr Ala Lys Val Thr Ser Ser Arg Gln Glu 65 70 75 80 Leu Asn Pro Leu LysSer Ile Val Glu Lys Ser Ile Leu Leu Thr Glu 85 90 95 Gln Ala Leu Ala LysAla Gly Lys Gly Met His Gly Gly Val Pro Gly 100 105 110 Gly Lys Gln PheIle Glu Asn Gly Ser Glu Phe Ala Gln Lys Leu Leu 115 120 125 Lys Lys PheSer Leu Leu Lys Pro Trp Ala 130 135 11 102 PRT Homo sapiens 11 Met LysLeu Ile Ala Leu Leu Phe Leu Thr Ala Leu Ala Gly Ala Leu 1 5 10 15 ValCys Ala Gln Asp Ala Pro Val Glu Glu Thr Pro Thr Glu Thr Pro 20 25 30 AlaGlu Thr Pro Ala Glu Thr Pro Ala Glu Thr Pro Ala Glu Thr Pro 35 40 45 AlaPro Ala Glu Ala Thr Gln Glu Thr Pro Ala Pro Ala Gln Glu Thr 50 55 60 ProAla Ala Thr Gln Ala Thr Ser Ala Ala Thr Gln Ala Thr Ser Ser 65 70 75 80Ile Thr Pro Ala Lys Ser Gly Ser Phe Leu Asp Gly Leu Lys Asn Lys 85 90 95Phe Lys Val Leu Leu Gly 100 12 20 PRT Homo sapiens 12 Ala Gly Glu AspPro Gly Leu Ala Arg Gln Ala Pro Lys Pro Arg Lys 1 5 10 15 Gln Arg SerSer 13 18 PRT Homo sapiens 13 Asp Ala Val Glu Asp Leu Glu Ser Val GlyLys Gly Ala Val His Asp 1 5 10 15 Val Lys

What is claimed is:
 1. A purified antibody that specifically binds to anepitope within the sequence of SEQ ID NO:5 (amino acids 20-42 ofpro-IBC-1).
 2. The antibody of claim 1, wherein the antibody is amonoclonal antibody.
 3. A purified antibody that specifically binds toan epitope within the sequence of SEQ ID NO:6 (amino acids 43-64 ofpro-IBC-1).
 4. The antibody of claim 3, wherein the antibody is amonoclonal antibody.
 5. The antibody of claim 3, wherein the epitope iswithin the sequence of SEQ ID NO:12 (amino acids 45-64 of pro-IBC-1). 6.The antibody of claim 5, wherein the antibody is a polyclonal antibody.7. The antibody of claim 5, wherein the antibody is a monoclonalantibody.
 8. The antibody of claim 3, wherein the epitope is within thesequence of SEQ ID NO:7 (amino acids 53-64 of pro-IBC-1).
 9. Theantibody of claim 8, wherein the antibody is a polyclonal antibody. 10.The antibody of claim 8, wherein the antibody is a monoclonal antibody.11. A purified antibody that specifically binds to an epitope within thesequence of SEQ ID NO:13 (amino acids 86-103 of pro-IBC-1).
 12. Theantibody of claim 11, wherein the antibody is a monoclonal antibody. 13.The antibody of claim 11, wherein the antibody is a polyclonal antibody.14. A method for determining whether a test sample comprises a receptorfor IBC-1, the method comprising: providing a test sample from a humanpatient; contacting the test sample with a polypeptide comprisingbetween 10 and 91 consecutive amino acids of IBC-1; and determiningwhether the polypeptide binds to the test sample; wherein detection ofthe polypeptide bound to the test sample in an amount higher than anegative control indicates that the test sample comprises a receptor forIBC-1.
 15. The method of claim 14, wherein the polypeptide is IBC-1. 16.The method of claim 14, wherein the test sample is prepared from abreast cancer, substantia nigra, pons, or hypothalamus tissue sample.17. The method of claim 14, wherein the patient is suspected of having,or being likely to develop, invasive and metastatic breast cancer. 18.The method of claim 14, wherein the patient is suspected of sufferingfrom or being at risk for developing a degenerative neural condition.19. A method for determining whether a patient has, or is likely todevelop, invasive and metastatic breast cancer, the method comprising:(a) providing a test sample from a breast cancer patient; and (b)detecting IBC-1 mRNA or IBC-1 in the test sample; wherein detection ofIBC-1 mRNA or IBC-1 in the test sample in an amount higher than in acontrol sample indicates that the patient has, or is likely to develop,invasive and metastatic breast cancer.
 20. The method of claim 19,wherein the test sample is prepared from a breast cancer tissue sample.21. The method of claim 19, wherein the test sample is prepared from abody fluid.
 22. A method for determining whether a patient has, or islikely to develop, invasive and metastatic breast cancer, the methodcomprising: (a) providing a test sample comprising genomic DNA from abreast cancer patient; and (b) determining whether genomic IBC-1 DNA isamplified in the test sample; wherein the presence of amplified genomicIBC-1 DNA in the test sample indicates that the patient has, or islikely to develop, invasive and metastatic breast cancer.
 23. The methodof claim 22, wherein the test sample is prepared from a breast cancertissue sample.
 24. A method for determining whether a patient issuffering from, or at risk for developing, a degenerative neuralcondition, the method comprising: (a) providing a test sample from ahuman patient suspected of suffering from, or being at risk fordeveloping, a degenerative neural condition; and (b) detecting IBC-1MRNA or IBC-1 in the test sample; wherein an amount of IBC-1 mRNA orIBC-1 in the test sample less than an amount of IBC-1 mRNA or IBC-1 in anormal control sample indicates that the patient is suffering from, orat risk for developing, a degenerative neural condition.
 25. The methodof claim 24, wherein the test sample is prepared from a substantianigra, pons or hypothalamus tissue sample.
 26. The method of claim 24,wherein the test sample is prepared from a body fluid.
 27. A method foridentifying a compound that blocks binding of IBC-1 to its receptor, themethod comprising: (a) providing a polypeptide comprising between 10 and91 consecutive amino acids of IBC-1, wherein the polypeptide binds anIBC-1 receptor; (b) providing a cell expressing the IBC-1 receptor; (c)contacting the cell with the polypeptide in the presence of a testcompound; and (d) determining whether the test compound blocks bindingof the polypeptide to the cell, as an indication that the compoundblocks binding of IBC-1 to its receptor.
 28. The method of claim 27,wherein the polypeptide is IBC-1.
 29. The method of claim 27, whereinthe cell is a breast cancer cell.
 30. The method of claim 27, whereinthe cell is a neural cell.
 31. The method of claim 27, wherein the testcompound is an antibody.
 32. The method of claim 31, wherein theantibody binds to IBC-1.
 33. The method of claim 31, wherein theantibody binds to the receptor.
 34. The method of claim 27, wherein thetest compound is a peptide.
 35. The method of claim 27, wherein the testcompound is a non-peptide small molecule.
 36. A process of making acompound, the process comprising: (a) carrying out the method of claim27 to identify a compound that blocks binding of IBC-1 to its receptor;and (b) manufacturing the compound.
 37. A method of treating cancer, themethod comprising: (a) identifying a patient having an invasive andmetastatic breast cancer that expresses IBC-1 or an IBC-1 receptor; and(b) treating the patient with a compound that blocks binding of IBC-1 toits receptor.
 38. The method of claim 37, wherein the compound is anantibody that binds to IBC-1.
 39. The method of claim 37, wherein thecompound is an antibody that binds to the receptor.
 40. The method ofclaim 37, wherein the patient is not diagnosed as suffering fromcachexia.
 41. The method of claim 37, wherein the patient does notexhibit overt symptoms of cachexia.
 42. A method of treating cancer, themethod comprising: (a) identifying a patient having an invasive andmetastatic breast cancer that expresses IBC-1 or an IBC-1 receptor; and(b) treating the patient with a compound that inhibits expression ofIBC-1 or its receptor.
 43. The method of claim 42, wherein the patientis not diagnosed as suffering from cachexia.
 44. The method of claim 42,wherein the patient does not exhibit overt symptoms of cachexia.
 45. Amethod of treating a neural condition, the method comprising: (a)identifying a patient suffering from, or at risk for developing, aneural condition involving degeneration of substantia nigra, pons orhypothalamus cells; and (b) administering IBC-1 or an IBC-1 agonist tothe patient.
 46. A kit comprising: (a) an agent for determining thelevel of IBC-1 in a biological sample; and (b) instructions for use ofthe agent for detecting invasive and metastatic breast cancer in apatient.
 47. A kit comprising: (a) an agent for determining whethergenomic IBC-1 DNA is amplified in a biological sample; and (b)instructions for use of the agent for detecting invasive and metastaticbreast cancer in a patient.